RESUMEN
Cellular processes including lysosomal and mitochondrial dysfunction are implicated in the development of many diseases. Quantitative visualization of mitochondria and lysosoesl is crucial to understand how these organelles are dysregulated during disease. To address a gap in live-imaging tools, we developed GEM-SCOPe (Genetically Encoded and Modular SubCellular Organelle Probes), a modular toolbox of fluorescent markers designed to inform on localization, distribution, turnover, and oxidative stress of specific organelles. We expressed GEM-SCOPe in differentiated astrocytes and neurons from a human pluripotent stem cell PRKN-knockout model of Parkinson's disease and identified disease-associated changes in proliferation, lysosomal distribution, mitochondrial transport and turnover, and reactive oxygen species. We demonstrate GEM-SCOPe is a powerful panel that provide critical insight into the subcellular mechanisms underlying Parkinson's disease in human cells. GEM-SCOPe can be expanded upon and applied to a diversity of cellular models to glean an understanding of the mechanisms that promote disease onset and progression.
RESUMEN
Inducible pluripotent stem cells (iPSCs) derived from patient samples have significantly enhanced our ability to model neurological diseases. Comparative studies of dopaminergic (DA) neurons differentiated from iPSCs derived from siblings with Gaucher disease discordant for parkinsonism provides a valuable avenue to explore genetic modifiers contributing to GBA1-associated parkinsonism in disease-relevant cells. However, such studies are often complicated by the inherent heterogeneity in differentiation efficiency among iPSC lines derived from different individuals. To address this technical challenge, we devised a selection strategy to enrich dopaminergic (DA) neurons expressing tyrosine hydroxylase (TH). A neomycin resistance gene (neo) was inserted at the C-terminus of the TH gene following a T2A self-cleavage peptide, placing its expression under the control of the TH promoter. This allows for TH+ DA neuron enrichment through geneticin selection. This method enabled us to generate comparable, high-purity DA neuron cultures from iPSC lines derived from three sisters that we followed for over a decade: one sibling is a healthy individual, and the other two have Gaucher disease (GD) with GBA1 genotype N370S/c.203delC+R257X (p.N409S/c.203delC+p.R296X). Notably, the younger sister with GD later developed Parkinson disease (PD). A comprehensive analysis of these high-purity DA neurons revealed that although GD DA neurons exhibited decreased levels of glucocerebrosidase (GCase), there was no substantial difference in GCase protein levels or lipid substrate accumulation between DA neurons from the GD and GD/PD sisters, suggesting that the PD discordance is related to of other genetic modifiers.
RESUMEN
Parkinson's disease (PD) is characterized pathologically by the loss of dopaminergic (DA) neurons in the substantia nigra (SN). Whether cell types beyond DA neurons in the SN show vulnerability in PD remains unclear. Through transcriptomic profiling of 315,867 high-quality single nuclei in the SN from individuals with and without PD, we identified cell clusters representing various neuron types, glia, endothelial cells, pericytes, fibroblasts, and T cells and investigated cell type-dependent alterations in gene expression in PD. Notably, a unique neuron cluster marked by the expression of RIT2, a PD risk gene, also displayed vulnerability in PD. We validated RIT2-enriched neurons in midbrain organoids and the mouse SN. Our results demonstrated distinct transcriptomic signatures of the RIT2-enriched neurons in the human SN and implicated reduced RIT2 expression in the pathogenesis of PD. Our study sheds light on the diversity of cell types, including DA neurons, in the SN and the complexity of molecular and cellular changes associated with PD pathogenesis.
Asunto(s)
Células Endoteliales , Enfermedad de Parkinson , Humanos , Animales , Ratones , Enfermedad de Parkinson/genética , Sustancia Negra , Neuronas Dopaminérgicas , NeuroglíaRESUMEN
Accumulation of advanced glycation end products (AGEs) on biopolymers accompanies cellular aging and drives poorly understood disease processes. Here, we studied how AGEs contribute to development of early onset Parkinson's Disease (PD) caused by loss-of-function of DJ1, a protein deglycase. In induced pluripotent stem cell (iPSC)-derived midbrain organoid models deficient for DJ1 activity, we find that lysosomal proteolysis is impaired, causing AGEs to accumulate, α-synuclein (α-syn) phosphorylation to increase, and proteins to aggregate. We demonstrated these processes are at least partly driven by astrocytes, as DJ1 loss reduces their capacity to provide metabolic support and triggers acquisition of a pro-inflammatory phenotype. Consistently, in co-cultures, we find that DJ1-expressing astrocytes are able to reverse the proteolysis deficits of DJ1 knockout midbrain neurons. In conclusion, astrocytes' capacity to clear toxic damaged proteins is critical to preserve neuronal function and their dysfunction contributes to the neurodegeneration observed in a DJ1 loss-of-function PD model.
Asunto(s)
Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/genética , Proteostasis , Astrocitos , Proteolisis , Mesencéfalo , Organoides , LisosomasRESUMEN
Functional output of the hippocampus, a brain region subserving memory function, depends on highly orchestrated cellular and molecular processes that regulate synaptic plasticity throughout life. The structural requirements of such plasticity and molecular events involved in this regulation are poorly understood. Specific molecules, including tissue inhibitor of metalloproteinases-2 (TIMP2) have been implicated in plasticity processes in the hippocampus, a role that decreases with brain aging as expression is lost. Here, we report that TIMP2 is highly expressed by neurons within the hippocampus and its loss drives changes in cellular programs related to adult neurogenesis and dendritic spine turnover with corresponding impairments in hippocampus-dependent memory. Consistent with the accumulation of extracellular matrix (ECM) in the hippocampus we observe with aging, we find that TIMP2 acts to reduce accumulation of ECM around synapses in the hippocampus. Moreover, its deletion results in hindrance of newborn neuron migration through a denser ECM network. A novel conditional TIMP2 knockout (KO) model reveals that neuronal TIMP2 regulates adult neurogenesis, accumulation of ECM, and ultimately hippocampus-dependent memory. Our results define a mechanism whereby hippocampus-dependent function is regulated by TIMP2 and its interactions with the ECM to regulate diverse processes associated with synaptic plasticity.
Asunto(s)
Encéfalo , Plasticidad Neuronal , Recién Nacido , Humanos , Plasticidad Neuronal/fisiología , Encéfalo/metabolismo , Neuronas/metabolismo , Hipocampo/metabolismo , Matriz Extracelular/metabolismo , Sinapsis/metabolismo , Inhibidor Tisular de Metaloproteinasa-2/genética , Inhibidor Tisular de Metaloproteinasa-2/metabolismoRESUMEN
The hypothalamus is a region of the brain that plays an important role in regulating body functions and behaviors. There is a growing interest in human pluripotent stem cells (hPSCs) for modeling diseases that affect the hypothalamus. Here, we established an hPSC-derived hypothalamus organoid differentiation protocol to model the cellular diversity of this brain region. Using an hPSC line with a tyrosine hydroxylase (TH)-TdTomato reporter for dopaminergic neurons (DNs) and other TH-expressing cells, we interrogated DN-specific pathways and functions in electrophysiologically active hypothalamus organoids. Single-cell RNA sequencing (scRNA-seq) revealed diverse neuronal and non-neuronal cell types in mature hypothalamus organoids. We identified several molecularly distinct hypothalamic DN subtypes that demonstrated different developmental maturities. Our in vitro 3D hypothalamus differentiation protocol can be used to study the development of this critical brain structure and can be applied to disease modeling to generate novel therapeutic approaches for disorders centered around the hypothalamus.
RESUMEN
Parkinson's disease (PD) is a complex neurodegenerative disease with etiology rooted in genetic vulnerability and environmental factors. Here we combine quantitative epidemiologic study of pesticide exposures and PD with toxicity screening in dopaminergic neurons derived from PD patient induced pluripotent stem cells (iPSCs) to identify Parkinson's-relevant pesticides. Agricultural records enable investigation of 288 specific pesticides and PD risk in a comprehensive, pesticide-wide association study. We associate long-term exposure to 53 pesticides with PD and identify co-exposure profiles. We then employ a live-cell imaging screening paradigm exposing dopaminergic neurons to 39 PD-associated pesticides. We find that 10 pesticides are directly toxic to these neurons. Further, we analyze pesticides typically used in combinations in cotton farming, demonstrating that co-exposures result in greater toxicity than any single pesticide. We find trifluralin is a driver of toxicity to dopaminergic neurons and leads to mitochondrial dysfunction. Our paradigm may prove useful to mechanistically dissect pesticide exposures implicated in PD risk and guide agricultural policy.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedades Neurodegenerativas , Enfermedad de Parkinson , Plaguicidas , Humanos , Plaguicidas/toxicidad , Enfermedad de Parkinson/genética , Neuronas DopaminérgicasRESUMEN
Cell state changes are associated with proteome remodeling to serve newly emergent cell functions. Here, we show that NGN2-driven conversion of human embryonic stem cells to induced neurons (iNeurons) is associated with increased PINK1-independent mitophagic flux that is temporally correlated with metabolic reprogramming to support oxidative phosphorylation. Global multiplex proteomics during neurogenesis revealed large-scale remodeling of functional modules linked with pluripotency, mitochondrial metabolism, and proteostasis. Differentiation-dependent mitophagic flux required BNIP3L and its LC3-interacting region (LIR) motif, and BNIP3L also promoted mitophagy in dopaminergic neurons. Proteomic analysis of ATG12-/- iNeurons revealed accumulation of endoplasmic reticulum, Golgi, and mitochondria during differentiation, indicative of widespread organelle remodeling during neurogenesis. This work reveals broad organelle remodeling of membrane-bound organelles during NGN2-driven neurogenesis via autophagy, identifies BNIP3L's central role in programmed mitophagic flux, and provides a proteomic resource for elucidating how organelle remodeling and autophagy alter the proteome during changes in cell state.
Asunto(s)
Células Madre Embrionarias Humanas/metabolismo , Proteínas de la Membrana/metabolismo , Mitocondrias/enzimología , Mitofagia , Células-Madre Neurales/enzimología , Neurogénesis , Neuronas/enzimología , Proteoma , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteína 12 Relacionada con la Autofagia/genética , Proteína 12 Relacionada con la Autofagia/metabolismo , Línea Celular , Humanos , Proteínas de la Membrana/genética , Mitocondrias/genética , Dominios y Motivos de Interacción de Proteínas , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Proteostasis , Proteínas Proto-Oncogénicas/genética , Factores de Tiempo , Proteínas Supresoras de Tumor/genéticaRESUMEN
Here, we describe a high-throughput 3D differentiation protocol for deriving midbrain dopaminergic neurons from human pluripotent stem cells. The use of organoids has become prevalent in disease modeling, but there is a high demand for more homogeneous cultures. Our approach is advantageous for large-scale production of uniform midbrain organoids that can be maintained in diverse formats, and our reporters allow for sorting of dopaminergic neurons. The maturing long-term organoid cultures can be used as a model for the entire midbrain. For complete details on the use and execution of this protocol, please refer to Ahfeldt et al. (2020).
Asunto(s)
Neuronas Dopaminérgicas , Mesencéfalo , Organoides , Células Madre Pluripotentes , Neuronas Dopaminérgicas/citología , Neuronas Dopaminérgicas/metabolismo , Humanos , Mesencéfalo/citología , Mesencéfalo/metabolismo , Organoides/citología , Organoides/metabolismo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismoRESUMEN
The derivation of human embryonic stem cells followed by the discovery of induced pluripotent stem cells and leaps in genome editing approaches have continuously fueled enthusiasm for the development of new models of neurodegenerative diseases such as Parkinson's disease (PD). PD is characterized by the relative selective loss of dopaminergic neurons (DNs) in specific areas of substantia nigra pars compacta (SNpc). While degeneration in late stages can be widespread, there is stereotypic early degeneration of these uniquely vulnerable neurons. Various causes of selective vulnerability have been investigated but much remains unclear. Most studies have sought to identify cell autonomous properties of the most vulnerable neurons. However, recent findings from genetic studies and model systems have added to our understanding of non-cell autonomous contributions including regional-specific neuro-immune interactions with astrocytes, resident or damage-activated microglia, neuro-glia cell metabolic interactions, involvement of endothelial cells, and damage to the vascular system. All of these contribute to specific vulnerability and, along with aging and environmental factors, might be integrated in a complex stressor-threshold model of neurodegeneration. In this forward-looking review, we synthesize recent advances in the field of PD modeling using human pluripotent stem cells, with an emphasis on organoid and complex co-culture models of the nigrostriatal niche, with emerging CRISPR applications to edit or perturb expression of causal PD genes and associated risk factors, such as GBA, to understand the impact of these genes on relevant phenotypes.
Asunto(s)
Células Madre Pluripotentes Inducidas , Enfermedad de Parkinson , Células Madre Pluripotentes , Neuronas Dopaminérgicas , Células Endoteliales , Humanos , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/terapia , Sustancia NegraRESUMEN
An increasing number of identified Parkinson's disease (PD) risk loci contain genes highly expressed in innate immune cells, yet their role in pathology is not understood. We hypothesize that PD susceptibility genes modulate disease risk by influencing gene expression within immune cells. To address this, we have generated transcriptomic profiles of monocytes from 230 individuals with sporadic PD and healthy subjects. We observed a dysregulation of mitochondrial and proteasomal pathways. We also generated transcriptomic profiles of primary microglia from brains of 55 subjects and observed discordant transcriptomic signatures of mitochondrial genes in PD monocytes and microglia. We further identified 17 PD susceptibility genes whose expression, relative to each risk allele, is altered in monocytes. These findings reveal widespread transcriptomic alterations in PD monocytes, with some being distinct from microglia, and facilitate efforts to understand the roles of myeloid cells in PD as well as the development of biomarkers.
Asunto(s)
Enfermedad de Parkinson , Humanos , Enfermedad de Parkinson/genética , Monocitos/metabolismo , Perfilación de la Expresión Génica , Transcriptoma , Encéfalo/metabolismoRESUMEN
The genetic architecture of each individual comprises common and rare variants that, acting alone and in combination, confer risk of disease. The cell-type-specific and/or context-dependent functional consequences of the risk variants linked to brain disease must be resolved. Coupling human induced pluripotent stem cell (hiPSC)-based technology with CRISPR-based genome engineering facilitates precise isogenic comparisons of variants across genetic backgrounds. Although functional-validation studies are typically performed on one variant in isolation and in one cell type at a time, complex genetic diseases require multiplexed gene perturbations to interrogate combinations of genes and resolve physiologically relevant disease biology. Our aim is to discuss advances at the intersection of genomics, hiPSCs and CRISPR. A better understanding of the molecular mechanisms underlying disease risk will improve genetic diagnosis, drive phenotypic drug discovery and pave the way toward precision medicine.
Asunto(s)
Encefalopatías/genética , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Genoma/genética , Humanos , Células Madre Pluripotentes Inducidas/fisiología , Medicina de Precisión/métodosRESUMEN
Parkinson's disease (PD) is a complex and highly variable neurodegenerative disease. Familial PD is caused by mutations in several genes with diverse and mostly unknown functions. It is unclear how dysregulation of these genes results in the relatively selective death of nigral dopaminergic neurons (DNs). To address this question, we modeled PD by knocking out the PD genes PARKIN (PRKN), DJ-1 (PARK7), and ATP13A2 (PARK9) in independent isogenic human pluripotent stem cell (hPSC) lines. We found increased levels of oxidative stress in all PD lines. Increased death of DNs upon differentiation was found only in the PARKIN knockout line. Using quantitative proteomics, we observed dysregulation of mitochondrial and lysosomal function in all of the lines, as well as common and distinct molecular defects caused by the different PD genes. Our results suggest that precise delineation of PD subtypes will require evaluation of molecular and clinical data.
Asunto(s)
Neuronas Dopaminérgicas/metabolismo , Genes Recesivos , Estudios de Asociación Genética , Predisposición Genética a la Enfermedad , Enfermedad de Parkinson/genética , Enfermedad de Parkinson/metabolismo , Transducción de Señal , Línea Celular , Técnicas de Sustitución del Gen , Humanos , Mitocondrias/metabolismo , Mutación , Enfermedad de Parkinson/diagnóstico , Fenotipo , Células Madre Pluripotentes/citología , Células Madre Pluripotentes/metabolismo , Proteoma , Proteómica/métodos , Tirosina 3-Monooxigenasa/genética , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
Flux through kinase and ubiquitin-driven signaling systems depends on the modification kinetics, stoichiometry, primary site specificity, and target abundance within the pathway, yet we rarely understand these parameters and their spatial organization within cells. Here we develop temporal digital snapshots of ubiquitin signaling on the mitochondrial outer membrane in embryonic stem cell-derived neurons, and we model HeLa cell systems upon activation of the PINK1 kinase and PARKIN ubiquitin ligase by proteomic counting of ubiquitylation and phosphorylation events. We define the kinetics and site specificity of PARKIN-dependent target ubiquitylation, and we demonstrate the power of this approach to quantify pathway modulators and to mechanistically define the role of PARKIN UBL phosphorylation in pathway activation in induced neurons. Finally, through modulation of pS65-Ub on mitochondria, we demonstrate that Ub hyper-phosphorylation is inhibitory to mitophagy receptor recruitment, indicating that pS65-Ub stoichiometry in vivo is optimized to coordinate PARKIN recruitment via pS65-Ub and mitophagy receptors via unphosphorylated chains.
Asunto(s)
Células Madre Embrionarias Humanas/enzimología , Membranas Mitocondriales/enzimología , Células-Madre Neurales/enzimología , Neurogénesis , Neuronas/enzimología , Proteómica/métodos , Ubiquitina-Proteína Ligasas/metabolismo , Activación Enzimática , Células HeLa , Humanos , Cinética , Mitofagia , Fenotipo , Fosforilación , Proteínas Quinasas/genética , Proteínas Quinasas/metabolismo , Transducción de Señal , Ubiquitina-Proteína Ligasas/química , Ubiquitina-Proteína Ligasas/genética , Ubiquitinación , Canal Aniónico 1 Dependiente del Voltaje/genética , Canal Aniónico 1 Dependiente del Voltaje/metabolismoRESUMEN
Utilizing patient derived cells has enormous promise for discovering new drugs for diseases of the nervous system, a goal that has been historically quite challenging. In this review, we will outline the potential of human stem cell derived neuron models for assessing therapeutics and high-throughput screening and compare to more traditional drug discovery strategies. We summarize recent successes of the approach and discuss special considerations for developing human stem cell based assays. New technologies, such as genome editing, offer improvements to help overcome the challenges that remain. Finally, human neurons derived from patient cells have advantages for translational research beyond drug screening as they can also be used to identify individual efficacy and safety prior to clinical testing and for dissecting disease mechanisms. This article is part of a Special Issue entitled SI: Exploiting human neurons.
Asunto(s)
Descubrimiento de Drogas , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Enfermedades del Sistema Nervioso/fisiopatología , Neuronas/fisiología , Animales , HumanosRESUMEN
Transcription activator-like effector nucleases (TALENs) are a new class of engineered nucleases that are easier to design to cleave at desired sites in a genome than previous types of nucleases. We report here the use of TALENs to rapidly and efficiently generate mutant alleles of 15 genes in cultured somatic cells or human pluripotent stem cells, the latter for which we differentiated both the targeted lines and isogenic control lines into various metabolic cell types. We demonstrate cell-autonomous phenotypes directly linked to disease-dyslipidemia, insulin resistance, hypoglycemia, lipodystrophy, motor-neuron death, and hepatitis C infection. We found little evidence of TALEN off-target effects, but each clonal line nevertheless harbors a significant number of unique mutations. Given the speed and ease with which we were able to derive and characterize these cell lines, we anticipate TALEN-mediated genome editing of human cells becoming a mainstay for the investigation of human biology and disease.
Asunto(s)
Desoxirribonucleasas/genética , Células Madre/enzimología , Alelos , Genoma Humano/genética , Humanos , MutaciónRESUMEN
Deriving lung progenitors from patient-specific pluripotent cells is a key step in producing differentiated lung epithelium for disease modeling and transplantation. By mimicking the signaling events that occur during mouse lung development, we generated murine lung progenitors in a series of discrete steps. Definitive endoderm derived from mouse embryonic stem cells (ESCs) was converted into foregut endoderm, then into replicating Nkx2.1+ lung endoderm, and finally into multipotent embryonic lung progenitor and airway progenitor cells. We demonstrated that precisely-timed BMP, FGF, and WNT signaling are required for NKX2.1 induction. Mouse ESC-derived Nkx2.1+ progenitor cells formed respiratory epithelium (tracheospheres) when transplanted subcutaneously into mice. We then adapted this strategy to produce disease-specific lung progenitor cells from human Cystic Fibrosis induced pluripotent stem cells (iPSCs), creating a platform for dissecting human lung disease. These disease-specific human lung progenitors formed respiratory epithelium when subcutaneously engrafted into immunodeficient mice.
Asunto(s)
Línea Celular , Fibrosis Quística , Células Madre Embrionarias , Células Madre Pluripotentes Inducidas , Pulmón , Células Madre Multipotentes , Animales , Línea Celular/metabolismo , Línea Celular/patología , Fibrosis Quística/metabolismo , Fibrosis Quística/patología , Células Madre Embrionarias/metabolismo , Células Madre Embrionarias/patología , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Células Madre Pluripotentes Inducidas/patología , Células Madre Pluripotentes Inducidas/trasplante , Pulmón/metabolismo , Pulmón/patología , Ratones , Ratones Endogámicos NOD , Ratones SCID , Células Madre Multipotentes/metabolismo , Células Madre Multipotentes/patología , Mucosa Respiratoria/metabolismo , Mucosa Respiratoria/patología , Transducción de Señal , Trasplante de Células Madre , Trasplante Heterólogo , Trasplante HomólogoRESUMEN
The utility of human pluripotent stem cells is dependent on efficient differentiation protocols that convert these cells into relevant adult cell types. Here we report the robust and efficient differentiation of human pluripotent stem cells into white or brown adipocytes. We found that inducible expression of PPARG2 alone or combined with CEBPB and/or PRDM16 in mesenchymal progenitor cells derived from pluripotent stem cells programmed their development towards a white or brown adipocyte cell fate with efficiencies of 85%-90%. These adipocytes retained their identity independent of transgene expression, could be maintained in culture for several weeks, expressed mature markers and had mature functional properties such as lipid catabolism and insulin-responsiveness. When transplanted into mice, the programmed cells gave rise to ectopic fat pads with the morphological and functional characteristics of white or brown adipose tissue. These results indicate that the cells could be used to faithfully model human disease.
Asunto(s)
Adipocitos Marrones/metabolismo , Adipocitos Blancos/metabolismo , Diferenciación Celular/genética , Células Madre Pluripotentes/metabolismo , Transgenes/genética , Células 3T3 , Adipocitos Marrones/citología , Adipocitos Blancos/citología , Tejido Adiposo Pardo/citología , Tejido Adiposo Pardo/metabolismo , Tejido Adiposo Blanco/citología , Tejido Adiposo Blanco/metabolismo , Animales , Proteína beta Potenciadora de Unión a CCAAT/genética , Proteína beta Potenciadora de Unión a CCAAT/metabolismo , Células Cultivadas , Análisis por Conglomerados , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Perfilación de la Expresión Génica , Células HEK293 , Humanos , Inmunohistoquímica , Trasplante de Células Madre Mesenquimatosas/métodos , Células Madre Mesenquimatosas/citología , Células Madre Mesenquimatosas/metabolismo , Ratones , Ratones Noqueados , Análisis de Secuencia por Matrices de Oligonucleótidos , PPAR gamma/genética , PPAR gamma/metabolismo , Células Madre Pluripotentes/citología , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Trasplante HeterólogoRESUMEN
Human pluripotent stem cells (hPSC) hold great promise as models for understanding disease and as a source of cells for transplantation therapies. However, the lack of simple, robust and efficient culture methods remains a significant obstacle for realizing the utility of hPSCs. Here we describe a platform for the culture of hPSCs that 1) allows for dissociation and replating of single cells, 2) significantly increases viability and replating efficiency, 3) improves freeze/thaw viability 4) improves cloning efficiency and 5) colony size variation. When combined with standard methodologies for genetic manipulation, we found that the enhanced culture platform allowed for lentiviral transduction rates of up to 95% and electroporation efficiencies of up to 25%, with a significant increase in the total number of antibiotic-selected colonies for screening for homologous recombination. We further demonstrated the utility of the enhanced culture platform by successfully targeting the ISL1 locus. We conclude that many of the difficulties associated with culturing and genetic manipulation of hPSCs can be addressed with optimized culture conditions, and we suggest that the use of the enhanced culture platform could greatly improve the ease of handling and general utility of hPSCs.